SUPPLEMENT ARTICLE

Facing the Crisis: Improving the Diagnosis

of Tuberculosis in the HIV Era
Mark D. Perkins1 and Jane Cunningham2
1
Foundation for Innovative New Diagnostics and 2United Nations Development Programme/United Nations Children’s Fund/World Bank/World
Health Organization Special Programme for Research and Training in Tropical Diseases (TDR), Geneva, Switzerland

Although the human immunodeficiency virus (HIV) infection pandemic has had a catastrophic impact on
tuberculosis (TB) control efforts, especially in sub-Saharan Africa, most of the fundamental concepts reflected
in the directly observed treatment, short course (DOTS) strategy still hold true in the HIV era. What has
changed, and dramatically, is the importance of speedy and accurate TB diagnosis and the difficulty of achieving
this. The disproportionate amount of smear-negative disease in sub-Saharan Africa, which shoulders two-
thirds of the global burden of HIV infection and acquired immunodeficiency syndrome, has greatly complicated
TB case detection and disease control. Now, 15 years after TB rates began to soar in countries where HIV
infection is prevalent, we have learned that the conventional approach—passively waiting for patients with
advanced symptomatic disease to make their way to microscopy centers for diagnosis—has disastrous con-
sequences. Without better diagnostic tools for TB and effective strategies for their implementation, transmission
will not be interrupted, mortality will not be checked, and TB will not be controlled in areas where HIV
infection is prevalent. Fortunately, a number of technical opportunities exist for the creation of improved
diagnostic tests. Developing and exploiting such tests to support TB control in HIV-infected populations is
an urgent priority. A substantial public sector effort is under way to work in partnership with the biotechnology
industry to accelerate progress toward that goal. In this article, we will define the need for better TB tests
and describe technologies being developed to meet that need.

BACKGROUND mens has remained the cornerstone of tuberculosis

HIV-mediated immunosuppression impairs granuloma
formation, resulting in both ineffective containment of
Mycobacterium tuberculosis bacilli and diminished for-
mation of pulmonary cavities [1, 2]. These effects man-
ifest clinically as frequent extrapulmonary disease [3],
atypical chest radiographic findings [4, 5], greater in-
volvement of the lower lobes of the lung, and lower
concentrations of bacteria in sputum [6].
Since Koch’s discovery of tuberculous bacilli in 1882,
microscopic detection of the bacilli in clinical speci-
Potential conflicts of interest: none reported.
Financial support: supplement sponsorship is detailed in the Acknowledgments.
Reprints or correspondence: Dr. Mark D. Perkins, Foundation for Innovative New
Diagnostics, 71 ave. Louis Casaı̈, PO Box 93, 1216 Cointrin, Switzerland (mark
.perkins@finddiagnostics.org).
The Journal of Infectious Diseases 2007; 196:S15–27
 2007 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2007/19604S1-0004$15.00
DOI: 10.1086/518656
(TB) diagnosis in low- and middle-income countries.
The failure to control TB in HIV-endemic areas has
underscored 2 major limitations to microscopic diag-
nosis: the low clinical sensitivity of the technique in
HIV-infected individuals and the logistic difficulty of
ensuring good access to quality microscopy in resource-
limited settings.
The first problem is sensitivity. In research settings
in which culture comparison is used, the average sen-
sitivity of sputum microscopy for the detection of pul-
monary TB is !60% in immunocompetent populations
[6–15] and is substantially lower among people infected
with HIV [16–19]. The actual sensitivity of microscopy
under field conditions may be much lower. The fraction
of HIV-coinfected individuals with pulmonary TB that
can be detected by microscopy varies widely with the
degree of immunosuppression, the length of TB illness,
and the local diagnostics setting, including the orga-
nization and strength of the TB control program and
its laboratory infrastructure.

Diagnostics for HIV-Associated TB • JID 2007:196 (Suppl 1) • S15

 The second problem is limited access to quality microscopy
services. The inherent low sensitivity of the test is too often
exacerbated by the poor conditions under which it is per-
formed. In overworked, underfunded laboratories, especially in
areas where HIV infection is prevalent, the proportion of cases
detected by microscopy is often as low as 20%–35% [20–23].
Duplicate or triplicate sputum examinations employed to help
overcome this problem—each of which requires sputum col-
lection, smear preparation, staining, and meticulous exami-
nation—delays results, and a relatively large number of patients
do not complete testing or are lost to the health care system
despite having tested positive for TB [24].
Limited access to diagnostic services results in substantial
diagnostic delay, and patients in many countries with a high
TB burden do not receive diagnoses for 3–6 months [25, 26].
This delay fuels disease transmission and increases the severity
of disease when it is finally discovered. This latter fact para-
doxically drives up the fraction of all patients with TB reported
as having smear-positive disease, which may be misinterpreted
as evidence of good performance of the microscopy network
when, in fact, it represents late detection and underdiagnosis
of smear-negative disease.
Frequent smear-negative disease exacerbates the difficulty of
detecting HIV-associated TB, leading to additional delays while
diagnostic testing or antibiotic treatment trials are being per-
formed. For example, more than a third of patients with smear-
negative TB in Malawi needed 16 visits to a health center before
therapy was initiated [27]. Examination of as many as 9 sputum
smears is recommended before reaching a diagnosis of smear-
negative TB in many countries [28]. The failure to rapidly detect
TB in immunocompromised populations has important im-
plications both for patient care and disease control. Although
the conventional wisdom is that smear-negative cases do not
contribute significantly to transmission, it is not known wheth-
er this holds true in populations in which HIV-associated im-
munosuppression is common. What is clear is that the failure
to detect TB early in HIV-coinfected individuals is lethal. Up
to 20% of all patients with TB who have treatment initiated in
sub-Saharan Africa die within a year [29], and two-thirds of
these deaths may occur in the first 2 months, which reflects
the advanced state of illness at the time of final diagnosis.
Whereas smear-negative TB has conventionally been regarded
as a slowly progressive disease with limited mortality, in HIV-
infected cohorts, patients with smear-negative disease often
have poorer treatment outcomes and greater mortality than do
their counterparts with smear-positive disease [30, 31].
The true magnitude of the problem of smear-negative TB in
severely immunocompromised individuals may be underesti-
mated, because many such patients may die before their TB is
detected. In Malawi, fully half of the patients with suspected
TB in whom a diagnosis was not readily made died while their
S16 • JID 2007:196 (Suppl 1) • Perkins and Cunningham
condition was under investigation [32]. Autopsy studies in
South Africa [33], the Ivory Coast [34], Botswana [35], the
Democratic Republic of the Congo [36], Kenya [37], and India
[38] have revealed TB as the leading cause of death in patients
who died with HIV coinfection. Such studies have also shown
that the accuracy of predeath diagnosis was poor [39, 40]. Thus,
HIV coinfection decreases the sensitivity of microscopy to de-
tect TB at the same time that it increases the need for rapid
diagnosis and treatment.
In response to the striking early mortality due to TB in
individuals with HIV coinfection and the poor performance of
microscopy, health care workers have been driven to use the
response to presumptive treatment as a diagnostic method [41].
The specificity of this approach is poor, leading to wasted drug
resources, overburdened treatment programs, and mistreat-
ment of many patients with other diseases. Mortality rates
among such mistreated patients are high [31].
DIAGNOSTIC PRIORITIES
Much is being done to identify HIV-associated TB more quickly
by implementing active case finding and abbreviating clini-
cal algorithms; clearly, however, better tests are needed. The
greatest need is for tests that can improve the detection of active
TB among symptomatic individuals. Better tests could abbre-
viate the period between the onset of symptoms and the ini-
tiation of therapy by being more sensitive, faster to yield results,
or simpler to use. An ideal test would combine these features
in a simple point-of-care format that could replace microscopy
and culture and yield a confirmatory diagnosis at the first clinic
visit. Such a test would, of course, have an important impact
on both HIV-infected and HIV-uninfected populations.
Improved detection of multidrug resistance (MDR) is also
needed. Global surveillance data, when adjusted for prior TB,
have not shown a close link between HIV infection and the
risk for multidrug-resistant TB (MDR-TB) [42]. However, in
some settings in developing countries, striking levels of HIV-
related MDR-TB have been found, often related to nosocomial
transmission through hospitals and clinics [43, 44]. Recent re-
ports, accompanied by substantial news coverage, of lethal out-
breaks of extensively drug-resistant TB (XDR-TB) in South
Africa and elsewhere have highlighted the need for rapid meth-
ods to identify highly resistant M. tuberculosis strains [45].
Lastly, improved detection of latent infection with M. tuber-
culosis in patients with HIV infection, with greater negative and
positive predictive values than current tuberculin skin testing,
could better target preventive therapy.
OPPORTUNITIES FOR BETTER DIAGNOSTICS
A summary of the technical opportunities for detecting HIV-
associated TB is presented below, focusing on case detection
technologies, drug susceptibility testing (DST) methods, and,
to a limited extent, tools for detecting latent infection (table
1). Methods of DST, which can be performed with a number
of case-detection technologies, are described in a separate
section.
Improvements in microscopy. Microscopic examination of
clinical specimens remains the most widely available test for
active TB and is the diagnostic centerpiece of the directly ob-
served treatment, short course (DOTS) strategy. Microscopy
has the advantages of being inexpensive, relatively rapid to
perform, and specific in most settings. Although microscopic
detection of acid-fast bacilli (AFB) in sputum is not specific
for TB in industrialized countries [46], the vast majority of
AFB-positive specimens in TB-endemic countries represent TB,
even in settings where nontuberculous mycobacteria may com-
monly be recovered in culture [8–15, 47].
Although AFB microscopy has changed little over the past
100 years, microscopy in other fields has evolved markedly.
Thus far, little has been done to harvest these advances for
improved TB diagnostics, and no successful automated mi-
croscopy system, for example, has thus far been developed. The
2 common alternatives to conventional direct microscopy that
have been adopted in some settings to improve speed or sen-
sitivity are fluorescence microscopy and alternative specimen-
processing methodologies.
Fluorescence microscopy, developed three-quarters of a cen-
tury ago for the detection of TB by exploiting the affinity of
fluorochromes (auramine and/or rhodamine) for mycolic acids
in the mycobacterial cell wall [48, 49], has been widely used
in industrialized countries to improve the speed and sensitivity
of AFB microscopy. By use of 25–40⫻ objectives, fluorescence
microscopy allows a much larger viewing field than is seen with
100⫻ oil immersion objectives used for carbol fuchsin–stained
slides. The sensitivity advantages of fluorescence over light mi-
croscopy for the detection of pulmonary TB have recently been
confirmed in a systematic review of 45 studies comparing the
2 methods, which found that fluorescence microscopy yielded
an average increase in sensitivity of 10%, with no loss of spec-
ificity [50]. There are few data evaluating fluorescence mi-
croscopy specifically in HIV-infected populations, but there is
little reason to believe that the findings would not be similar.
Publicly supported field evaluations of various fluorescent mi-
croscopy systems are under way to determine performance and
feasibility.
Equipment costs limit the wider use of fluorescence micro-
scopes. The microscopes may cost between $10,000 and $20,000
and use an intense ultraviolet light source, such as a high-
pressure mercury lamp, which typically costs $100–$300 and
lasts only 100–200 h. Light-emitting diodes, which have a life
span of 110,000 h and replacement costs that may be less than
$5, can also excite dyes such as auramine and rhodamine to
fluoresce but have suffered from being considerably dimmer
than mercury lamps. Ultrabright light-emitting diodes 30–50
times brighter than standard light-emitting diodes have been
developed, and preliminary data have demonstrated the utility
of such microscope systems for TB detection [51].
Most microscopic examinations for pulmonary TB are per-
formed directly on smeared and stained preparations of un-
processed sputum, and the search for sputum-processing meth-
ods that could improve the yield, safety, or ease of microscopy
is many decades old [52]. The poor performance of TB mi-
croscopy for individuals with HIV coinfection has given new
urgency to this area of work. A variety of chemical and physical
methods have been used either to concentrate bacilli by means
of gravitational force or filtration or to improve their dispersion
in sputum. A recent systematic review of the literature in this
area identified 83 studies examining the comparative yield of
an alternative method to direct microscopy. The majority of
studies that used any procedure for digestion/liquefaction fol-
lowed by centrifugation, prolonged gravity sedimentation, or
filtration [53] found an increase in sensitivity of 13%–33% over
direct microscopy, when culture was used as the reference stan-
dard [54]. Unfortunately, given the variability in patient selec-
tion and trial design between published studies, it is not possible
to determine which, if any, of these methods is optimal. The
easy availability of household bleach (5% sodium hypochlorite),
along with its microbicidal activity, make it a commonly studied
processing reagent, and multicenter studies to determine the
feasibility and impact of wider implementation of a standard-
ized bleach-processing method are planned [55].
Growth-based detection. Mycobacterial culture on selec-
tive media remains the most sensitive method for detecting M.
tuberculosis bacilli in clinical specimens and allows subsequent
strain characterization, including DST. The slow replication
time of M. tuberculosis requires that solid media cultures be
incubated for 2–8 weeks (depending on the inoculated bacterial
concentration) for the growth of the millions of organisms
necessary to generate visible colonies. This process can be ac-
celerated by microscopically detecting immature colonies, de-
tecting products of bacterial replication, or using bacterio-
phages as markers of mycobacterial viability. Several tests have
been developed to exploit these principles.
Automated liquid culture systems have been developed to
accompany conventional solid media culture. These systems
detect bacterial CO2 production or O2 consumption with ra-
diometric, fluorescent, colorimetric, or pressure sensors that
allow continuous monitoring, obviate the need for mature col-
ony formation, and roughly halve the time to detection, com-
pared with Löwenstein-Jensen culture [56–66]. Because of the
expense of the sophisticated culture vials employed, the usual
need for large and expensive incubator/readers, and the rec-
ommended requirement for additional backup culture on solid
media, these culture systems have seen limited use in TB-en-
Diagnostics for HIV-Associated TB • JID 2007:196 (Suppl 1) • S17
Table 1. Tuberculosis (TB) diagnostic technologies, stage of development, and settings of use.

Level of the DST

Technology, test Stage of development Developer(s)/supplier(s) health systema utilityb

Case detection
Growth-based detection
Conventional solid mediac Commercialized reagents and Multiple Referral Y
prepared media
Automated liquid culture systems Commercialized, under study BD, bioMérieux, Trek Referral Y
for feasibility and impact of
use in resource-limited
settings
TK colorimetric media In evaluation Salubris Referral Y
MODS assay, thin-layer culture, Academic evaluations Noncommercial testing Referral Y
others published methods
Phage-based detection Commercialized, improved Biotec Referral Y
test in development
Direct visualization
Conventional microscopy with In routine use Multiple Microscopy N
acid-fast staining
Fluorescent microscopy with In routine use Multiple Microscopy N
nonspecific cell-wall staining
Fluorescent microscopy with LED In development Various Microscopy N
light source
Fluorescent microscopy with In development ID-FISH Technology Microscopy N
molecular probes (FISH)
Automated microscopy In development Various Microscopy N
Computer-assisted microscopy In development Various Microscopy N
VOC detection
Electronic nose analysis of In development Scensive Microscopy N
headspace gas
GC/MS analysis of exhaled air In development Menssana Research Microscopy N
Handheld surface acoustic In development Electronic Sensor Technology Microscopy N
wave-GC
Giant African pouch rats In evaluation Apopo Microscopy N
Honeybees In development Inscentinel Microscopy N
Antigen detection
TB-derived antigen detection in In development Chemogen, Proteome Systems, Health center N
urine or other clinical material TB DiaDirect, others
TB-derived antigen detection in In evaluation Rapid Biosensor Systems Health center N
exhaled air vapor
Antibody detection
Detection of diagnostic antibody Many commercially Various Health center N
responses to TB available, improved
tests in development
Molecular detection
Automated, nonintegrated NAAT Commercialized GenProbe, Roche, BD, others Reference N
Automated, integrated NAAT In development Cepheid Referral Y
Simplified manual NAAT (LAMP) In development Eiken Microscopy N
Nonamplified probe detection In development Investigen, others Microscopy N
Transrenal DNA detection In development Xenomics, others Referral or microscopy N
Manual amplification and In evaluation Innogenetics, Hain Reference N
hybridization

(continued)
Table 1. (Continued.)

Level of the DST

Technology, test Stage of development Developer(s)/supplier(s) health systema utilityb
d
Species identification
Luminescent probe of culture isolate Commercially available GenProbe Reference N
Fluorescent probe of smear-positive In development ID-FISH Referral N
sputum
Reverse hybridization line probe Commercially available Innogenetics, Hain Reference N
from culture isolates
Dipstick detection of TB antigens in In demonstratione Tauns Referral N
positive cultures
Species-specific amplification or Research use Various Reference N
sequencing
LTBI detection
Tuberculin skin test with PPD Commercialized Multiple Microscopy N
MPT-64 skin patch In evaluation Sequella Health center N
Whole-blood IFN-g release assay Commercialized; in evaluation Cellestis Referral N
for disease-endemic
countries
ELISPOT IFN-g release assay Commercialized; in evaluation Oxford Immunotech Referral N
for disease-endemic
countries
Skin testing with TB-specific In early evaluation Statens Serum Institut Microscopy N
antigens

NOTE. DST, drug-susceptibility testing; ELISPOT, enzyme-linked immunospot; FISH, fluorescence in situ hybridization; GC, gas chromatography; IFN, interferon;
LAMP, loop-mediated, isothermal amplification; LED, light-emitting diode; LTBI, latent TB infection; MODS, microscopic-observation drug susceptibility assay;
MS, mass spectrometry; NAAT, nucleic acid amplification testing; PPD, purified protein derivative; VOC, volatile organic compound.
a
The health care system is divided here for convention into 4 levels: reference laboratory, a national or regional laboratory performing specialty mycobacterial
tests, not focused on patient care; referral laboratory, a laboratory with TB-specific expertise performing such tests as TB culture; microscopy laboratory, a
laboratory performing only microscopy for TB detection; and health center, a clinical facility not routinely providing any mycobacteriology testing. Listed in the
table is the level of intended or appropriate use.
b
Indicates that methodology may also be used to detect drug resistance.
c
Löwenstein-Jensen, Ogawa, 7H10, and other media.
d
Beyond NAATs with species-specific primer sequences.
e
Demonstration is a phase in FIND’s development pathway, coming after evaluation, in which the feasibility and impact of programmatic use are measured.

demic countries. The Foundation for Innovative New Diag-
nostics (FIND) is working in collaboration with the Consor-
tium to Respond Effectively to the AIDS/TB Epidemic (CREATE)
and Becton Dickinson to execute large-scale demonstration
projects of liquid culture in Zambia, South Africa, and Brazil.
These projects will examine the feasibility, impact, and cost-
effectiveness of using cost-reduced Mycobacteria Growth In-
dicator Tube (MGIT; Becton Dickinson) culture for the detec-
tion of TB in settings of high HIV infection prevalence.
Potential disadvantages of liquid culture include a high risk
of contamination, especially when used by laboratories not ex-
perienced in liquid culture of TB, and the lack of colony mor-
phology examination as a protection against unexpected growth
of non-TB mycobacteria. A solid media system called “TK,”
which is being developed by Salubris in partnership with FIND,
uses a proprietary media formulation that allows colorimetric
detection of bacterial growth before the appearance of visible
colonies [67]. Additionally, contaminating nonmycobacterial
species cause a different color shift, to green rather than red,
which allows simple discrimination between AFB-positive and
contaminated vials. Preliminary studies reported that the TK
media system, which is relatively inexpensive to manufacture,
detected TB 10 days faster than did conventional Löwenstein-
Jensen media in a limited number of cultures [68]. Multicenter
evaluations to confirm the performance of TK media have yet
to be performed.
Noncommercial methods to speed mycobacterial detection
have also been developed. A variety of redox reagents, such as
Alamar blue [69], have been used to detect early growth in
liquid culture but have not seen widespread use. Similarly, mi-
croscopic examination of thin-layer agar plates for the early
detection of mycobacterial microcolonies [70] has been pro-
posed, as has microscopic examination of liquid culture. By
adding antituberculous antibiotics to adjacent wells and ex-
amining for comparative growth, this latter technique (termed
the “microscopic-observation drug susceptibility assay”) has

Diagnostics for HIV-Associated TB • JID 2007:196 (Suppl 1) • S19

been applied for early detection of drug resistance as well, as
is described below in the “DST” section [71].
The ability of mycobacteriophages to infect and replicate in
viable M. tuberculosis has been exploited in diagnostic assays.
In one, a lytic mycobacteriophage, D29, is used to infect M.
tuberculosis bacilli in processed sputum after a short incubation
period in media. After the infection period, a virucide is added
that kills exogenous phage while not affecting phage internal-
ized within the mycobacteria. Infected cells, once plated on a
lawn of the rapidly growing Mycobacterium smegmatis, produce
plaques indicating the presence of TB. This biological ampli-
fication procedure can be performed in days, compared with
weeks for culture, and has theoretically similar sensitivity. This
has been developed commercially as the FASTPlaqueTB assay
(Biotec Laboratories). In its current form, the test detects 29%–
87% of smear-positive disease cases and 13%–78% of smear-
negative disease cases within 2 days [72–77]. The capacity of
D29 phage to replicate in nontuberculous mycobacteria has not
impaired clinical specificity, which remained high (99.1%) even
in a study in which 30% of all culture isolates were nontu-
berculous mycobacteria [74]. Biotec has partnered with FIND
to work toward the development of a more sensitive version
of the assay. Although the test is laboratory based and involves
multiple steps, this assay may offer an alternative to nucleic
acid amplification testing (NAAT) for the rapid detection of
smear-negative TB at a cost similar to that of culture on com-
mercial media.
The superior performance of culture is accompanied by a
range of technical and logistical obstacles that are particularly
relevant in the developing world. With the exception of Brazil,
the Russian Federation, and South Africa, culture facilities are
rare in countries with a high TB burden [78]. Therefore, broadly
implementing culture or phage replication as a solution to the
problem of smear-negative TB will require significant invest-
ment in laboratory infrastructure.
Antigen detection. Attempts have been made to detect M.
tuberculosis antigens in body fluids, as an alternative to con-
ventional microbiological confirmation. The detection of pro-
tein and nonprotein antigens has demonstrated diagnostic util-
ity for a number of other diseases, including malaria, influenza,
and bacterial meningitis, but no such test for TB has been
successfully commercialized. Theoretical advantages to this ap-
proach include quantitative correlation with burden of disease,
the likelihood of high specificity, and lack of dependence on a
functioning immune response. Assays using serum or urine
might have particular application in HIV-associated TB, in
which extrapulmonary disease is common and a large total
body burden of bacilli may not be reflected in the sputum.
Some attempts at antigen detection for TB diagnosis have
used polyclonal antibodies against whole cell or other crude
antigen preparations to detect M. tuberculosis antigens in blood,
S20 • JID 2007:196 (Suppl 1) • Perkins and Cunningham
cerebrospinal fluid, or urine [79–82]. More recently, monoclo-
nal antibodies have been developed to target proteins and gly-
colipids abundant in culture filtrate, such as MPT32 [83], the
antigen 85 complex [84–86], the 38 kDa protein [85, 87], and
lipoarabinomannan. Lipoarabinomannan is a heat-stable, high-
molecular-weight glycolipid present in the lipophilic cell well
of mycobacteria in a variety of forms in and across different
mycobacterial species. Several groups have demonstrated mea-
surable concentrations of lipoarabinomannan in the sputum
[88, 89], serum [90], and urine [91–95] of patients with TB.
FIND is partnering with industry and academic researchers to
develop a sensitive and specific commercial lipoarabinomannan
detection assay for clinical use.
The only commercial antigen detection test for TB is an assay
using the M. tuberculosis–specific antigen MPB-64 (Tauns) as
a target for mycobacterial species identification. This simple
lateral flow test allows accurate differentiation of M. tuberculosis
complex organisms isolated in solid or liquid culture from the
remainder of the mycobacterial species. Rapid species identi-
fication of culture isolates, most often accomplished using ex-
pensive molecular probes, will be important to support the
increased use of culture for TB detection in areas where HIV
infection is prevalent [96–99]. Unfortunately, the product is
currently not available for purchase outside of Japan. FIND is
working to make this test available for the public sector in
developing countries.
Almost all work on antigen detection has targeted secreted
or cell surface antigens present in culture filtrate. Other anti-
gens, expressed in vivo, may also have diagnostic value. Ex-
ploratory work at Proteome Systems [100] has identified a
number of novel proteins, not found in culture filtrate, that
can be detected at low concentrations in body fluids of patients
with TB. Additional work in this area may yield reagents tar-
geting an antigen or series of antigens that could be used to
develop a point-of-care TB antigen detection test.
Molecular detection. Aside from microscopy and culture,
the only proven method for detection of M. tuberculosis that
has been successfully developed as a clinical diagnostic tool is
NAAT. These tests use oligonucleotide primers and enzymes to
catalyze reiterative reactions that amplify a target, probe, or
signal, yielding a result within minutes to hours. The analytic
sensitivity of these systems tends to be exquisite, although clin-
ical performance may vary. Several TB NAAT methods have
been developed, and 2 are currently commercialized in the
United States. The 3 most widely used of these assays, poly-
merase chain reaction (PCR; Roche Diagnostics), transcription-
mediated amplification (GenProbe), and strand-displacement
amplification (Becton Dickinson) have shown excellent spec-
ificity and speed, as well as sensitivity approaching but not
equaling that of dual-media culture [101, 102]. Despite the clear
advantages of NAATs over existing tests, especially for the rapid
detection of smear-negative disease, they have only limited use
in TB-endemic settings, primarily because of their cost and
complexity. Testing itself is usually performed on expensive,
precision instruments that are beyond the capacity of most
diagnostic sites to purchase or maintain and that require skilled
technologists to operate. Even in established molecular labo-
ratories in resource-limited countries, performance is highly
variable, suggesting that much greater assay robustness or much
more laboratory support will be needed before NAAT can be
implemented more widely [103].
A number of groups have recently investigated methods to
simplify sample processing and molecular amplification. Biode-
fense spending has been an important impetus for this, as has
been the market for point-of-care testing in industrialized
countries. One exciting technology under development for the
detection of TB is the loop-mediated, isothermal amplification
(LAMP) method, invented by researchers at Eiken Chemical.
The advantages of this technology, which uses 6 specifically
designed primers and a single polymerase with strand-displace-
ment activity, are that it requires no thermocycler, is a closed
system, and gives a visual readout interpretable by the naked
eye [104]. If sufficiently simple to use, such a test might be
implemented in microscopy centers to replace or augment mi-
croscopy to improve the sensitivity of case detection at this
level of the health system. Working with FIND, Eiken is de-
veloping a prototype test for TB, using a simplified specimen-
processing method that might feasibly be implemented in re-
source-limited areas. Preliminary data suggest that, in its
current form, the assay may be performed at the benchtop by
technicians with no molecular training and performs similarly
to commercialized instrumented systems, detecting essentially
all smear-positive specimens and half of smear-negative spec-
imens [105].
The requirement for specimen processing and DNA extrac-
tion is an important obstacle to the implementation of any
molecular amplification method in laboratories without sub-
stantial technical infrastructure. FIND is working with Cepheid
to develop a real-time PCR assay for TB on its GeneXpert
platform that automates sputum processing, DNA extraction,
gene amplification, and target detection into a single, hands-
free test. The assay, which is being developed in collaboration
with the University of Medicine and Dentistry of New Jersey,
will use molecular beacons to detect the presence of both TB
and rifampin resistance in !2 h [106]. The first clinical trials
of the test started in May 2007.
The finding that DNA from apoptotic cells in the body make
their way into the urine in short fragments (150–200 bp) in
predictable and detectable concentrations [107] has led to the
development of real-time PCR assays, for pregnancy-related,
cancer-related, and transplant-related diseases, that use urine
rather than invasively collected material. Testing for transrenal
DNA from M. tuberculosis has been performed in a preliminary
study of 20 patients with TB and TB-negative control subjects.
In that study, TB-specific DNA was found in all 20 patients
with TB and in none of the control subjects. The results of
urine culture for M. tuberculosis were negative [108]. The fre-
quency of extrapulmonary disease and paucibacillary pulmo-
nary disease among patients with HIV-associated TB may limit
the sensitivity of any sputum-based tests, so urine-based de-
tection of mycobacterial antigens or DNA is highly relevant for
this population. Further development of this approach, and
fieldwork to evaluate its potential utility in resource-limited
areas, is under way.
Diagnostic humoral immune responses. Many TB proteins
and nonprotein molecules are highly immunogenic. Exploiting
the humoral and cellular responses that they generate for a TB
diagnostic test is attractive, in part because of the potential
simplicity of testing in a dipstick or similar format. Immu-
nologic testing holds the additional theoretical advantage of
not depending on access to the infecting bacteria and, thus,
detecting extrapulmonary and paucibacillary disease as readily
as cavitary pulmonary TB.
Although responses are heterogeneous, antibodies to M. tu-
berculosis antigens can be detected in the majority of immu-
nocompetent patients with active TB. The development of se-
rologic tests for TB based on the detection of such antibodies
has been attempted for decades. For years, this work centered
on the 38 kDa antigen and a limited number of other antigens
abundant in culture filtrate or immunodominant in animals.
Although 12 dozen serologic tests based on these reagents are
currently being marketed, primarily in developing countries,
none of the existing commercial tests shows adequate sensitivity
and specificity for recommended use. Given the altered humoral
immune responses in patients with HIV-associated immuno-
compromise, it is not surprising that the performance of these
tests is especially poor in HIV-coinfected patients [109–111].
The failure of existing antibody-based TB tests to meet clin-
ical needs does not mean that development of such a tool is
not possible. Recent work to look beyond the targets commonly
used in current commercial kits has identified a number of
promising antigens that show high specificity on initial screen-
ing and might be usefully included in a multiantigen assay [112,
113]. More work is needed in 2 areas. First, a systematic dis-
covery effort is needed to identify additional targets among the
nearly 4000 proteins encoded by the M. tuberculosis genome,
particularly those not found in culture filtrate. Protein expres-
sion patterns of bacteria vary with the growth conditions of
the organism, including in response to immunologic pressure
[114], and it is likely that a number of antigens expressed during
different stages or types of infection will be needed for a test
that is sensitive in diverse clinical settings. For example, despite
the general blunting of antibody responses to TB in individuals
Diagnostics for HIV-Associated TB • JID 2007:196 (Suppl 1) • S21
with HIV coinfection [115], some antigens, such as TB9.7 and
81/88 kDa, may be preferentially expressed in individuals with
HIV coinfection and have specific diagnostic utility in that
population [112, 116]. This points to the second need, which
is for greater attention to the breadth and precise nature of
disease represented in the collections of clinical samples used
to evaluate antigens. Often, antigens are selected or evaluated
using serum from populations that are demographically re-
stricted, manifest only specific types of disease (e.g., smear-
positive pulmonary TB), or are poorly characterized clinically
and microbiologically.
Diagnostic immune responses. HIV-related immunocom-
promise is the most significant risk factor for reactivation TB
in individuals with latent TB infection (LTBI). The standard
tool, the tuberculin skin test, has a number of limitations, in-
cluding the need for injection, the subjectivity of readout, and
the cross-reactivity of purified protein derivative with bacille
Calmette-Guérin and other mycobacteria.
Two promising new in vitro tests of cell-mediated immunity
use M. tuberculosis–specific antigens encoded by DNA sequences
that have been deleted from all bacille Calmette-Guérin vaccine
strains and are not present in most species of nontuberculous
mycobacteria. QuantiFERON-TB Gold (Cellestis) uses a whole-
blood assay to measure interferon (IFN)–g production from
sensitized T cells, and the T SPOT-TB test (Oxford Immunotec)
uses an enzyme-linked immunospot assay to quantify the num-
ber of peripheral blood mononuclear cells producing IFN-g in
response to antigen stimulation. Both of these assays give ob-
jective results, with sensitivity, as measured in patients with
active TB, comparable to that of the tuberculin skin test. The
lack of cross-reactivity with bacille Calmette-Guérin gives im-
portant benefits in specificity, and, in low-incidence settings,
both tests give results that more closely associate with the degree
of clinical exposure to TB than does the tuberculin skin test.
Interesting preliminary data suggest that quantitative responses
to ESAT-6 might even be useful in detecting incipient disease
in individuals with LTBI [117]. A review of these technologies
has recently been published [118].
Whether these IFN-g assays will show adequate sensitivity
in HIV-infected populations to warrant their routine use in
endemic settings is an unresolved question, and early field data
are promising but conflicting [119, 120]. Although there is
also some interest in the use of these tests to rule out TB in
symptomatic patients with suspected TB, existing data suggest
that, even in largely HIV-uninfected populations, a significant
portion of patients with confirmed TB may be test negative
[121]. More operational research is needed to clarify the role
of these important new diagnostic methods for the control of
TB in resource-limited areas, especially where HIV infection is
prevalent.
Sensing volatile organic compounds and other biomarkers.
S22 • JID 2007:196 (Suppl 1) • Perkins and Cunningham
There is a growing interest in detecting TB by “sniffing” for
the presence of characteristic volatile compounds or patterns
of compounds in exhaled air or headspace gas over sputum or
bacterial cultures. Press reports have highlighted the efforts to
use animals, such as the African giant pouch rat (Cricetomys
gambianus), to detect TB [122]. Engineered sensing technol-
ogies that physically detect volatile organic compounds would
be much easier to implement. One such technology is the elec-
tronic nose, so called because it mimics a biological olfactory
system by using nonspecific gas sensors and pattern recognition
algorithms to detect and analyze thousands of different odors
with a relatively small number of nonselective receptors [123].
Currently, there are US Food and Drug Administration–ap-
proved applications for electronic noses in the detection of
urinary tract infection and bacterial vaginosis. Other health
applications are under development [124], including for de-
tection of TB, for which there are interesting preliminary data.
Fend et al. [125], using a nonoptimized sensor array of 14
conductive polymer sensors, were able to differentiate M. tu-
berculosis, Mycobacterium avium, and Pseudomonas aeruginosa
in headspace gas over spiked sputum. The analytic sensitivity
for M. tuberculosis was 104 cfu/mL. The clinical sensitivity values
were 91% and 89% in patient samples that were culture positive
(n p 55) or culture negative (n p 79 ), respectively, for M. tu-
berculosis. Interestingly, natural and experimental infection with
Mycobacterium bovis could also be detected in cattle and badgers
by sampling the headspace gas over serum [126].
More quantitative analytic approaches to volatile organic
compound detection have been used as well. In a recent study,
gas chromatography/mass spectroscopy analysis of exhaled air
has been used to distinguish patients with pulmonary TB from
healthy subjects and those with other types of lung disease, by
using pattern recognition and fuzzy logic [127]. The volatile
organic compounds recognized in the breath of patients with
TB were structurally similar to those found in headspace gas
over TB culture.
DRUG SUSCEPTIBILITY TESTING
Disease control efforts in TB-endemic countries have tradi-
tionally focused on case detection and not on DST. Ten years
ago, the phenomenon of MDR-TB gained attention as high-
prevalence foci in developing countries were recognized, prompt-
ing the expansion of DOTS treatment strategies to cover MDR-
TB (DOTS Plus). Recent recognition of clusters of rapidly fatal
cases of XDR-TB has, in a similar way, underscored the need
for expanded diagnostic strategies under DOTS.
In most developing countries, DST, where available, is usually
performed on solid media, such as Löwenstein-Jensen. Results
often come back after 8–18 weeks of waiting, during which
time many patients with MDR-TB or XDR-TB may have died,
transmitted their disease, or both. There are a number of re-
cently developed methods for the detection of drug resistance
that are faster, although usually no less complex, than conven-
tional DST on solid media. Some of these technologies are
described below. Unfortunately, the laboratory infrastructure
to support such testing in disease-endemic countries is very
limited. Of the 22 highest-TB-burden countries, fewer than half
have 13 laboratories in the entire national laboratory network
with the capacity of performing DST [128]. Thus, efforts to
contain drug-resistant TB must focus both on the development
of simpler and more rapid detection methods and on the im-
provement of laboratory capacity.
Growth-based methods of TB detection can usually also be
applied to test drug susceptibility, and many of the techniques
described above have been successfully adapted for DST [129–
135]. Over the past 2 decades, automated liquid culture systems
have come into common use in developed countries for DST
with both first- and second-line TB drugs, yielding results in
2–4 weeks. Performing DST in these systems directly from
smear-positive specimens can further reduce delays in receiving
results to 1–3 weeks [56, 136–139]. Noncommercial methods
for more rapid DST have also been published, including direct
solid media inoculation and readout via colony inspection or
by the addition of colorimetric substrates, as in the nitrate
reductase assay, or Greiss method [140]. The microscopic-ob-
servation drug susceptibility assay, mentioned briefly above, is
a noncommercial system based on microscopic observation of
early colony formation in liquid media. This inexpensive
method yields culture and drug resistance phenotype results in
just over a week and has a high correlation with DST using
reference methods, especially for rifampin and isoniazid [141].
The feasibility of programmatic implementation of such a
method, which is not available in a kit format, has not yet been
demonstrated, and its use is currently restricted to research
settings.
A version of the phage replication assay that can detect rif-
ampin resistance in 48 h directly from smear-positive sputum
has recently been launched commercially (FASTPlaque-Re-
sponse) and is under evaluation for use in the public sector of
resource-limited countries [142]. Genetically engineered lucif-
erase reporter phages have also been developed for rapid DST
[143–145]. This latter approach is not currently exploited in a
commercial assay and has the disadvantage of requiring a period
of culture before testing.
The restricted number of genetic mutations responsible for
rifampin resistance has been exploited by 2 currently available
commercial molecular assays, GenoType MTBDR (Hain Life-
Science) and INNO-LiPA.Rif TB (Innogenetics), both of which
use conventional PCR followed by amplicon hybridization onto
a series of oligonucleotide probes on nitrocellulose strips to
detect rifampin resistance with a 1-day turnaround. A number
of trials with these tests, performed on culture isolates or di-
rectly on smear-positive sputum, have shown good correlation
with conventional rifampin susceptibility testing [146–153].
The GeneXpert system described above [106] uses similar gene
targets but, by automating the processing, amplification, and
detection steps, aims to greatly simplify molecular testing, mak-
ing point-of-care TB detection and broad access to drug resis-
tance screening possible.
SUMMARY
In summary, there are a number of exciting technologies being
developed for the improved diagnosis of TB, including HIV-
associated TB. In the very short term, better case detection can
be achieved through revised diagnostic algorithms, improved
microscopy, and implementation of rapid mycobacterial cul-
ture. In the longer term, completely novel approaches are likely
to be available. Both short-term and long-term solutions, if
they are to have an impact, will require sustained support of
public-private partnerships coupled with a national commit-
ment to improve laboratory infrastructure and rapidly adopt
technologies with demonstrated utility.
Acknowledgments
Supplement sponsorship. This article was published as part of a sup-
plement entitled “Tuberculosis and HIV Coinfection: Current State of
Knowledge and Research Priorities,” sponsored by the National Institutes
of Health Division of AIDS, the Centers for Disease Control and Prevention
Division of TB Elimination, the World Bank, the Agence Nationale de
Recherches sur le Sida et les Hépatites Virales, and the Forum for Collab-
orative HIV Research (including special contributions from the World
Health Organization Stop TB Department, the International AIDS Society,
and GlaxoSmithKline).
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